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Callus Culture and Regeneration
Jaideep Mathur and Csaba Koncz
1. Introduction
Regeneration of plants by micropropagation of h vitro cultures can be
achieved from organ primordia existing in shoot tips and axillary bud explants.
Alternatively, plants can be regenerated from unorganized callus tissues derived
from different explants by dedifferentiation induced by exogenous growth
regulators. Plant regeneration from calli is possible by de novo organogenesis
or somatic embryogenesis. Callus cultures also facilitate the amplification of
limiting plant material. In addition, plant regeneration from calli permits the
isolation of rare somaclonal variants which result either from an existing
genetic variability in somatic cells or from the induction of mutations,
chromosome aberrations, and epigenetic changes by the in vitro applied
environmental stimuli, including growth factors added to the cultured cells (1-3).
In Arabidopsis thaliana, one of the earliest studies on callus formation was
conducted by Loewenberg (4), who grew seedlings in a medium containing
kinetin and parachlorophenoxyacetic acid. Subsequent studies (for review, see
ref. 5) of cell culture and regeneration demonstrated that it is relatively easy to
regenerate Arabidopsis from callus cultures by many approaches. However,
the efficiency of regeneration and the proportion of somaclonal variants are
considerably influenced by the ecotype and the source of explants, as well as
by the medium and growth regulators employed. Hypocotyl and root explants
thus provide excellent materials for callus initiation and regeneration in contrast
to stem and leaf explants. The tissue culture protocols include callus induction
in auxin containing media. Shoot regeneration is induced by lowering the auxin
content and increasing the cytokinin levels in the media. The elongated shoots
are usually transferred into root induction media, but even in the absence of
roots, flowering and seed setting can take place in test tubes in vitro.
From: Methods in Molecular Biology, Vol. 82: Arabidopsis Protocols
Edited by: J. Martinez-Zapater and J. Salinas Q Hurnana Press Inc.. Totowa. NJ
Mathur and Koncz
The method described here utilizes root explants that can be cultured and
efficiently regenerated in large quantities. The described technique can be
applied equally well for the Arabidopsis ecotypes Columbia, C24, RLD, and
Wassilewskija. Without changing the general protocol, some slight alterations
may be required for other ecotypes or mutants of Arabidopsis.
2. Materials
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Dry seeds of Arabidopsis thaliana.
10% Sodium hypochlorite solution containing 0.1% Triton X-100.
Eppendorf tubes.
Sterilized double-distilled water.
Culture tubes.
9 cm Petri dishes.
Pair of forceps.
Scissors or scalpel blades.
Sterile 250-mL Erlenmeyer flasks with plugs.
Rotary shaker set at 120 rpm.
Microfuge.
Basal medium (BM): Murashige and Skoog (MS) medium (6) or MS medium for
Arabidopsis (MSAR) medium (7) with 3% sucrose (pH 5.8) can equivalently be
used (see Note 1).
0.5X BM: BM consisting of half concentration of MS macroelements and
0.5% sucrose.
MSAR I (Callus medium): Supplement BM with 0.5 mg/L 2,4--dichlorc~
phenoxyacetic acid (2,4-D), 2.0 mg/L indole3-acetic acid (IAA), 0.5 m g L
6-(y,y-dimethylallylamino~purine riboside (IPAR), and gel using either
0.8% agar or 0.2% gelrite (see Note 1).
MSAR I1 (Shoot medium): Supplement BM with 2.0 mg/L 6-(y,y-dimethylallylamino)-purine riboside (IPAR), 0.05 mg/L a-naphtaleneacetic acid (NAA),
and either 0.8% agar or 0.2% gelrite.
MSAR 111 (root inducing medium): Supplement BM with 1.0 mg/L IAA,
0.2 mg/L indole3-butyric acid (IBA), 0.2 mg/L 6-furfuryl-aminopurine
(kinetin), and either 0.8% agar or 0.2% gelrite.
3. Methods
1. Surface sterilize 0.1 g (approx 5000) seeds in Eppendorf tubes by adding a 1 mL
of 10% (v/v) solution of sodium hypochlorite containing 0.1% Triton X-100 as
surfactant and shaking for about 15 min (see Note 2).
2. Pellet the seeds by slow centrifugation in a microfuge for a few seconds and
remove the supernatant. Wash the seeds five times with 1 mL of sterile water.
3. Germinate the seeds in Petri dishes containing 0.8% agar gelled 0.5X BM
medium (BM medium containing half concentration of macroelements)
using 16 h light and 8 h dark period at 25°C (see Note 3).
4. Place 15-20 1-wk-old seedlings into 250-mL Erlenmeyer flasks containing
approx 35 mL of liquid BM medium with 3% sucrose.
Callus Culture and Regeneration
5. Place the flasks on a rotary shaker at 120 rpm using 16 h light and 8 h dark
period at 25OC.
6. Harvest the roots of these seedlings after 15-20 d of growth (see Note 4).
7. Place the roots into a Petri dish, remove all liquid and cut the roots into small pieces.
8. Transfer root explants onto MSAR I plates and either cover them with aluminium
foil or place them in low light conditions.
9. After 3 wk, transfer the root-derived calli either to fresh MSAR I plates for
maintenance or to MSAR I1 plates for shoot regeneration.
10. After 2 wk, pick the regenerating shoots from the calli and transfer them into
glass jars with MSAR I1 medium for further elongational growth (see Note 5).
I 1. Shoots consisting of 4-6 leaves may be transferred further into MSAR 111medium
for 3-6 d to induce root development.
12. Transfer the elongated shoots (approx 2 cm) into culture tubes containing
0.5 BM agar medium with 0.5% sucrose for rooting, flowering, and setting seeds
(see Notes 6 and 7).
4. Notes
1. Stocks of growth regulators are prepared at 1 mg/mL concentration, filter
sterilized, and stored at 4OC. Growth regulators are added to autoclaved medium
after it has cooled to about 60°C.
2. Do not sterilize too many seeds in one Eppendorf tube, as they may not be easily
dried. In case seeds clump, use a sterile toothpick to break the clumps before
plating the seeds.
3. Calli can also be obtained from germinating seeds that are placed on MSAR I
plates after surface sterilization.
4. After 15-20 d of culture, 3-5 g (fresh weight) of roots should be produced in each
flask. The roots should be white, actively growing, and not yellow-brown or green.
5. At this stage, it is possible to regenerate and amplify shoots from the regenerated
leaves by cutting them into pieces and placing them on MSAR I1 Medium.
6. The culture tubes are capped with loose cotton to facilitate the aeration required
for seed setting and maturation. Take care not to place the tubes too close to the
light source because this will cause moisture condensation inside the tubes
and result in a low rate of fertilization.
7. Rooted plants can be transferred to soil after washing the roots with water
containing a fungicide, such as Benomyl (0.02%). The plants are gradually
acclimated by reducing the humidity of transfer chambers stepwise.
References
1. Flick, C. E., Evans, D. A., and Sharp, W. R. (1983) Organogenesis, in Handbook
ofplant Cell Culture, vol. 1 (Evans, D. A., Sharp, W. R., Amrnitato, P. V., and
Yamada, Y. eds.), MacMillan, New York, pp. 13-8 1.
2. Raghavan, V. (1986) Embryogenesis in Angiosperms. Cambridge University
Press, Cambridge, UK.
3. Larkin, P. J. and Scowcraft, W. R. (1981) Somaclonal variation - a novel
source of variability from cell culture for plant improvement. Theor. Appl. Genet.
60, 197-214.
Mathur and Koncz
4. Loewenberg, J. R. (1965) Callus cultures ofArabidopsis. Arabidopsis Inf: Sen. 2,34.
5 . Monis, P. C. and Altmann, T. (1994) Tissue culture and transformation, in Arabidopsis. (Meyerowitz, E. M. and Somerville, C. R., eds.), Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York, pp. 173-222.
6. Murashige, T. and Skoog, F. (1962) A revised medium for growth and bioassays
with tobacco tissue cultures. Physiol. Plant. 15,473497.
7. Koncz, C., Martini, N., Szabados, L., Hrouda, M., Bachmair, A., and Schell, J.
(1994) Specialized vectors for gene tagging and expression studies, in Plant
Molecular Biology Manual. (Gelvin, S. B . and Schilperoort, R. A., eds.), B2,
Kluwer, Dordrecht, pp. 1-22.